Congenital myasthenic syndromes (CMS) are characterized by fatigable muscle weakness resulting from impaired neuromuscular transmission. β2-adrenergic agonists are an effective treatment for DOK7-CMS. DOK7 is a component within the AGRN-LRP4-MUSK-DOK7 signalling pathway that is key for the formation and maintenance of the synaptic structure of the neuromuscular junction (NMJ). The precise mechanism of action of β2-adrenergic agonists at the NMJ is not fully understood. In this study, we investigated whether β2-adrenergic agonists improve both neurotransmission and structural integrity of the NMJ in a mouse model of DOK7-CMS. Ex-vivo electrophysiological techniques and microscopy of the NMJ were used to study the effect of salbutamol, a β2-adrenergic agonist, on synaptic structure and function. DOK7-CMS model mice displayed a severe phenotype with reduced weight gain and perinatal lethality. Salbutamol treatment improved weight gain and survival in DOK7 myasthenic mice. Model animals had fewer active NMJs, detectable by endplate recordings, compared with age-matched wild-type littermates. Salbutamol treatment increased the number of detectable NMJs during endplate recording. Correspondingly, model mice had fewer acetylcholine receptor-stained NMJs detected by fluorescent labelling, but following salbutamol treatment an increased number were detectable. The data demonstrate that salbutamol can prolong survival and increase NMJ number in a severe model of DOK7-CMS.

Background and Objectives Up to 50% of patients with myasthenia gravis (MG) without acetylcholine receptor antibodies (AChR-Abs) have antibodies to muscle-specific kinase (MuSK). Most MuSK antibodies (MuSK-Abs) are IgG4 and inhibit agrin-induced MuSK phosphorylation, leading to impaired clustering of AChRs at the developing or mature neuromuscular junction. However, IgG1-3 MuSK-Abs also exist in MuSK-MG patients, and their potential mechanisms have not been explored fully. Methods C2C12 myotubes were exposed to MuSK-MG plasma IgG1-3 or IgG4, with or without purified agrin. MuSK, Downstream of Kinase 7 (DOK7), and βAChR were immunoprecipitated and their phosphorylation levels identified by immunoblotting. Agrin and agrin-independent AChR clusters were measured by immunofluorescence and AChR numbers by binding of ¹²⁵I-α-bungarotoxin. Transcriptomic analysis was performed on treated myotubes. Results IgG1-3 MuSK-Abs impaired AChR clustering without inhibiting agrin-induced MuSK phosphorylation. Moreover, the well-established pathway initiated by MuSK through DOK7, resulting in βAChR phosphorylation, was not impaired by MuSK-IgG1-3 and was agrin-independent. Nevertheless, the AChR clusters did not form, and both the number of AChR microclusters that precede full cluster formation and the myotube surface AChRs were reduced. Transcriptomic analysis did not throw light on the pathways involved. However, the SHP2 inhibitor, NSC-87877, increased the number of microclusters and led to fully formed AChR clusters. Discussion MuSK-IgG1-3 is pathogenic but seems to act through a noncanonical pathway. Further studies should throw light on the mechanisms involved at the neuromuscular junction.

Recent work putatively linked a rare genetic variant of the chaperone Resistant to Inhibitors of acetylcholinesterase (RIC3) (NM_024557.4:c.262G > A, NP_078833.3:p.G88R) to a unique ability to speak backwards, a language skill that is associated with exceptional working memory capacity. RIC3 is important for the folding, maturation, and functional expression of α7 nicotinic acetylcholine receptors (nAChR). We compared and contrasted the effects of RIC3G88R on assembly, cell surface expression, and function of human α7 receptors using fluorescent protein tagged α7 nAChR and Förster resonance energy transfer (FRET) microscopy imaging in combination with functional assays and ¹²⁵I-α-bungarotoxin binding. As expected, the wild-type RIC3 protein was found to increase both cell surface and functional expression of α7 receptors. In contrast, the variant form of RIC3 decreased both. FRET analysis showed that RICG88R increased the interactions between RIC3 and α7 protein in the endoplasmic reticulum. These results provide interesting and novel data to show that a RIC3 variant alters the interaction of RIC3 and α7, which translates to decreased cell surface and functional expression of α7 nAChR.

Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10–25 mM) reduced endplate potential (EPP) amplitudes by 10–40% and enhanced depression during repetitive (2–20 Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole‐cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC₅₀of 3.74 mM. Cyclohexanol (10–20 mM) also caused prolonged episodes of reduced‐current, multi‐channel bursting in outside‐out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (−6 kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α‐subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K⁺currents. Modelling indicated cyclohexanol binding (−3.8 kcal/mol) to voltage‐sensitive K⁺channels at the same site as tetraethylammonium (TEA). TEA (10 mM) blocked K⁺channels more effectively than cyclohexanol but EPPs were more prolonged in 20 mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs.Key points: Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite. Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content). Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K⁺‐channel function. The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following intentional ingestion of agricultural organophosphorus insecticides. Our findings also extend understanding of the effects of alcohols on synaptic transmission and homeostatic synaptic function.

The muscle nicotinic acetylcholine receptor (AChR) is the key mediator of neuromuscular signal transmission and is essential for all voluntary movement in our body. In this study, we present DC-98-LC74, a positive allosteric modulator (PAM) for the adult skeletal muscle-type AChR. Through using Ca ²⁺ fluorometric imaging plate reader (FLIPR) assays, we demonstrate that it is selective for the adult skeletal muscle AChR over neuronal subtypes. Neurophysiological recordings from ex vivo mouse diaphragm preparations revealed that DC-98-LC74 elongates the endplate currents of wildtype (WT) adult but not fetal channel containing diaphragms. Single channel studies on chimeric channels of the adult and fetal receptor, and in saturating concentrations of choline, suggest that the PAM does not bind at either orthosteric site, but works by increasing the unliganded open probability via a mechanism that involves the ε M2-M3 loop. We also show that DC-98-LC74 increases the burst duration of multiple fast channel mutant AChR to WT levels, suggesting that positive allosteric modulation could be a therapeutic strategy for this difficult to treat subtype of congenital myasthenia. Promising preliminary data on aged sarcopenic mice also demonstrate that positive allosteric modulation of the muscle type AChR has potential benefits not only in myasthenia but also other neuromuscular disorders involving the neuromuscular junction.